Triterpenes from the aerial parts of Cimicifuga yunnanensis and their antiproliferative effects on p53N236S mouse embryonic fibroblasts

Article abstract of DOI:10.1021/np4000262

Nine new triterpene derivatives, yunnanterpenes A-F (1-6), 15,16-seco-cimiterpenes A and B (7, 8), and cimilactone C (9), and 15 known analogues (10-24) were isolated from the aerial parts of Cimicifuga yunnanensis. The new structures were established using a combination of MS, NMR, and single-crystal X-ray diffraction techniques. WT MEFs (wild-type mouse embryonic fibroblasts) and tumorigenic cell lines p53^-/-+H-RasV12 and p53 ^-/-+p53^N236S+H-RasV12 were used for evaluating active structures, targeting p53^N236S (corresponding to p53^N239S in humans) mutation. Compound 5 showed nonselective activities against these cell lines, with IC₅₀ values of 5.8, 8.6, and 6.0 μM, respectively. Compound 4 exhibited greater selectivity against the p53 ^-/-+p53^N236S+H-RasV12 cells (IC₅₀ 5.5 μM) than against the WT MEFs cells (IC₅₀ 14.3 μM).

Full text of DOI:10.1021/np4000262

Article
pubs.acs.org/jnp
Triterpenes from the Aerial Parts of Cimicifuga yunnanensis and Their
N236S
Antiproliferative Effects on p53
Mouse Embryonic Fibroblasts
†
‡
‡
‡
§
,†
Yin Nian, Hui Zhu, Wen-Ru Tang, Yin Luo, Jiang, Du, and Ming-Hua Qiu*
†State Key Laboratory of Phytochemistry, Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650201, People’s Republic of China
‡
Laboratory of Molecular Genetics of Aging & Tumor, Faculty of Medicine, Kunming University of Science & Technology, Kunming,
6
50500, People’s Republic of China
§
Research Institute of Yunnan Biovalley, Dengzhanhua Pharmaceutical Co. Ltd, Kunming, 650224, People’s Republic of China
S Supporting Information
*
ABSTRACT: Nine new triterpene derivatives, yunnanterpenes
A−F (1−6), 15,16-seco-cimiterpenes A and B (7, 8), and
cimilactone C (9), and 15 known analogues (10−24) were
isolated from the aerial parts of Cimicifuga yunnanensis. The new
structures were established using a combination of MS, NMR,
and single-crystal X-ray diffraction techniques. WT MEFs (wild-
type mouse embryonic fibroblasts) and tumorigenic cell lines
−
/−
−/−
N236S
p53 +H-RasV12 and p53 +p53
+H-RasV12 were used
N236S
for evaluating active structures, targeting p53
to p53
(corresponding
in humans) mutation. Compound 5 showed
N239S
nonselective activities against these cell lines, with IC50 values
of 5.8, 8.6, and 6.0 μM, respectively. Compound 4 exhibited
−/−
N236S
greater selectivity against the p53 +p53
+H-RasV12 cells
(
IC50 5.5 μM) than against the WT MEFs cells (IC50 14.3 μM).
9
current strategy in cancer treatment is to apply
personalized treatment aimed at different p53 mutants.
pathway. However, there have been no reports on the
1
A
chemical constituents of the aerial parts of C. yunnanensis.
Thus, we initiated a study on the aerial parts of C. yunnanensis
from Daocheng County. As a result, nine new triterpenes,
yunnanterpenes A−F (1−6), 15,16-seco-cimiterpenes A and B
(7, 8), and cimilactone C (9), and 15 known compounds (10−
24) were isolated and identified (Supporting Information,
Figure 1S). All of these compounds were evaluated for their
The IARC TP53 database (version R14, November 2009)
N239S
N236S
shows that human p53
(corresponding to p53
in the
mouse) has been reported as a somatic mutation in 32 tumor
cases and tumor origin tissues including breast, colon, stomach,
hematopoietic and reticuloendothelial systems, liver and
2
intrahepatic bile ducts, bronchus and lung, and brain. The
N239S
−/−
wide tumor spectrum harboring p53
suggested its
antiproliferative effects on the WT MEFs, p53 +H-RasV12,
and p53− +p53
/−
N236S
+H-RasV12 cell lines by the MTT
importance in tumorigenesis. Previous investigations on the
N236S
gained functions of the mutation indicated that p53
per se
method. Described herein are the isolation, structure
elucidation, and biological activities of these compounds.
N236S
was not oncogenic enough to form tumors. However, p53
can interact with H-RasV12 and reduce the cellular stress
response to oncogenic signals to facilitate cell growth and
RESULTS AND DISCUSSION
1
■
tumorigenesis. Thus, using nontumorigenic and p53 knockout
−
/−
Compound 1 was obtained as colorless crystals. The HREIMS
of 1 gave an [M] ion peak at m/z 502.3287 (calcd 502.3294),
consistent with the molecular formula C H O , requiring
eight rings or sites of unsaturation. The IR spectrum showed
absorptions for OH (3383 cm ) and carbonyl (1706 cm )
groups. The H NMR spectrum (Table 1) showed character-
istic cyclopropane methylene signals at δ 0.47 and 0.72 (each
H, d, J = 4.1 Hz), a secondary methyl signal at δ 1.46 (d, J =
mouse embryonic fibroblasts (cell line p53
MEFs) as
+
−
/−
background, H-RasV12 was introduced into p53 MEFs with
N236S
−/−
or without p53
, and two tumorigenic cell lines, p53 +H-
30 46
6
−/−
N236S
RasV12 and p53 +p53
+H-RasV12, were established for
−1
−1
N236S
evaluation of active structures, targeting p53
mutation.
1
As a part of our program to explore potential antitumor
constituents from traditional Chinese medicine, a series of
cytotoxic 9,19-cycloartane-type triterpenes were isolated from
Cimicifuga spp. (Ranunculaceae), such as C. foetida, C.
H
1
6
H
.5 Hz), and five tertiary methyl groups at δ 0.82−1.76. The
H
3
−8
yunnanensis, C. dahurica, and C. heracleifolia.
Three active
compounds isolated from the roots of C. yunnanensis induced
Received: January 16, 2013
apoptosis of MCF-7 cells via the p53-dependent mitochondrial
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
dx.doi.org/10.1021/np4000262 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
CH -27 (δ 1.76) and H-26α (δ 4.10). However, it was not
3
H
H
possible to identify the stereochemistry of C-23 by ROESY
experiment. To confirm the structure and determine the
absolute configuration of C-23, 1 was crystallized from MeOH
to afford a crystal of the orthorhombic space group P2 2 2 ,
1
1 1
which was analyzed by X-ray crystallography. On the basis of six
oxygen atoms in the molecule, the final refinement of the Cu
Kα data resulted in a Flack parameter of 0.06(11) and allowed
unambiguous assignment of the absolute configuration (Figure
2
). According to the projection, the absolute configuration at
C-23 was S. Therefore, the structure of 1, named
yunnanterpene A, was determined to be as shown.
Yunnanterpene B (2) and yunnanterpene C (3) were
determined to have the molecular formulas C H O and
3
0
48
6
+
C H O by HREIMS (m/z 504.3444 [M] and 546.3557
M] , respectively). The spectroscopic data of 2 (Tables 1 and
32
50
7
+
[
2
) were similar to those of 1 except that the carbonyl group was
replaced by a hydroxy group at C-3 in 2. The deduction was
confirmed by HMBC correlations of H-1 (δ 1.55 and 1.24)
H
and H-2 (δ 1.95 and 1.83) with C-3 (δ 77.52), as well as a
H
C
COSY correlation of H-2/H-3. The diagnostic ROESY
correlation of H-3/H-5 suggested the β-orientation of the
OH group at C-3. In addition, the same relative configurations
at C-16, C-17, C-20, C-24, and C-25, as well as the
conformations of rings E and F in 2 as in 1, were deduced
from the similar NMR chemical shifts, proton coupling
13C NMR and DEPT (Table 2) spectra displayed signals for
one carbonyl group, seven quaternary carbons (including two
oxygenated ones), seven methines (including three oxygenated
ones), nine methylenes (including an oxygenated one), and six
methyls. The data suggested that 1 was a highly oxygenated
constants, and ROESY correlations (H-16/CH -28, H-17/
3
CH -28 H-22β, H-26β/H-24, and H-26α/CH -27) found in 2.
3
3
Thus, the structure of 2 was established as shown, and it was
named yunnanterpene B. When the spectroscopic data of 3
9
,19-cycloartane-type triterpene, and a seven-ring structure was
required to fulfill the unsaturation requirement.
1
1
(
Tables 1 and 2) were compared with those of 2, the resonance
The H− H COSY (Figure 1) and HMQC spectra disclosed
that 1 had partial structures −CH −CH − (for C-1 to C-2),
of a hydroxy group was absent in 3, showing instead an O-
acetyl group functionality at C-12. The deduction was
2
2
−
CHCH CH CH− (for C-5 to C-8), −CH CH(OH)− (for
2
2
2
confirmed by the correlation of H-12 (δ 5.11) with the acetyl
C-11 to C-12), and −CH CHCHCH(CH )CH − (for C-15 to
H
2
3
2
carbonyl carbon at δ 170.57, as well as correlations of H-11
C-17, C-20 to C-22) and a pair of geminal signals for CH -26 at
C
2
1
1
(
δ 2.75 and 1.15) with H-12 in the H −H COSY spectrum.
Significant ROESY correlations of H-12 with H-17 and CH -28
δH 4.10 and 4.32 (each 1H, d, J = 8.6 Hz) that were typical of
H
rings A, B, C, D, and part of E and F of a 9,19-cycloartane-type
3
22
indicated β-orientation of the substituent at C-12. Accordingly,
the structure of 3 was determined as shown.
triterpene skeleton. HMBC correlations observed from H-26
at δ 4.10 and 4.32 to the two quaternary carbons at δ 110.87
H
C
Yunnanterpene D (4) was assigned the molecular formula
(
(
C-23) and 79.78 (C-25), from the methyl resonance at δ 1.76
H
+
C H O , as determined by HREIMS (m/z 504.3441 [M] ).
Me-27) to the quaternary carbon resonance at δ 79.78 (C-
30 48
6
C
The NMR data of compounds 4 and 2 were very similar, with
the main differences being that the signals ascribed to the
characteristic cyclopropane methylene were absent. However,
an additional methyl group was observed. On the basis of the
evidence, we deduced that the 9,19-cyclopropane ring of 4 was
open and the methyl group was attached at C-9 or C-10. In the
2
5), the methylene carbon resonance at δ 77.19 (C-26), and
C
the methine resonances at δ 83.67 (C-24), and from H-16 at
δH 4.71 (dd, J = 14.7, 7.8 Hz) to the quaternary carbon at δC
C
22
1
10.87 (C-23) were similar to those of 23-epi-26-deoxyactein
and indicated that rings E and F of 1 each contained one
oxygen unit. Since compound 1 had a carbonyl group (δC
13
2
14.97), no more rings were required. Thus, we deduced that
C NMR spectrum (Table 2), two quaternary olefinic carbons
OH groups were attached to C-24 and C-25 in 1. This
at δ 134.31 and 137.56 were observed. HMBC correlations of
C
conclusion was supported by the molecular weight, as well as by
H-7 (δH 1.88 and 1.09) and CH -28 (δH 0.85) with the
3
13
the signals of C-24 at δ 83.67 and 79.78 in the C NMR
unsaturated carbon signal at δ 134.31 (C-8) and of H-11 (δH
C
C
spectrum of 1. In the HMBC spectrum, the correlations of H-2
2.64 and 2.07) with the unsaturated carbon signal at δ 137.56
C
(
δ 1.43 and 1.71) and CH -30 (δ 1.12) with the carbonyl
(C-9) located the double bond at C-8 and C-9. Thus, the
H
3
H
carbon at δ 214.97 placed the carbonyl group at C-3. An OH
methyl group (CH -19) was attached at C-10, which was
further supported by the HMBC correlation of the methyl
C
3
1
1
was located at C-12 by the H− H COSY correlations of H-12
at δ 4.06 and H-11 at δ 2.57 and 1.48.
signal at δ 0.92 with C-1 at δ 36.59. The orientation of CH -
H
H
H
C
3
The relative configuration of the core structure of 1 was
19 was assigned as β by the ROESY correlation of CH -19 (δH
3
established by the ROESY experiment (Figure 1). The cross-
0.92) and CH -18 (δH 1.05). Thus, the structure of 4 was
3
peaks of H-12, H-16, and H-17 with CH -28 in the ROESY
determined as shown.
3
spectrum assigned their orientations as α. In addition, H-22β
Yunnanterpene E (5) was determined to have the molecular
+
(
δ 2.85) and H-26β (δ 4.32) showed correlations with H-24,
formula C H O by HREIMS (m/z 486.3344 [M] ). The UV
H
H
30 46
5
indicating an α-orientation of the OH group at C-24. The
and IR spectra of 5 showed absorption bands for hydroxy
groups (νmax 3425 cm ) and a unit of conjugated double
−1
orientation of CH -27 was assigned as α by the correlation of
3
B
dx.doi.org/10.1021/np4000262 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 1. H NMR Data of Compounds 1−9 (δ in ppm, J in Hz)
b
b
b
b
b
c
b
b
b
position
1
1
2
3
4
5
6
7
8
9
a
a
1.71 m
1.55 m
1.47 m
1.55 m
2.01 m
1.57 dd (14.2, 1.58
.5)
1.61
1.21 dd (13.2,
4.0)
3
a
a
a
a
a
1
.43
1.24
1.04
1.91 m
1.09 m
1.72 m
1.48 m
1.96 m
1.33
1.29 m
1.32 m
0.80
2
2.62 m
.28 m
1.95 m
2.35 m
2.34 brd (9.9) 2.32 brd (9.6) 1.67 m
a
a
2
1.83 dd (12.3, 1.80 m
.4)
1.12
1.22
1.90 qd (12.9, 1.91 m
4.0)
1.89 t (13.8)
1.56 m
3
3
3.52 m
3.47 m
3.32 m
3.48 m
3.49 dd (11.7, 3.49 dd (11.6, 3.49 dd (11.6, 3.24 dd (11.6,
4
.2)
3.8)
4.1)
3.7)
4
5
a
a
a
a
1.56 dd (12.1, 1.28 m
.1)
1.21
1.02 brd
(12.4)
1.24 dd (11.9, 1.55
5.0)
1.49 m
1.51
0.96
4
6
1.37 m
1.55 m
0.83 m
1.51 m
1.59 m
2.11 m
2.04 m
1.29 m
0.85 m
1.38 m
0.91 m
1.27 m
a
a
a
0.84
0.77 dd (24.6, 1.40 m
2.3)
1.22
1.33
0.51 dd (25.3,
12.8)
1
a
a
7
1.30 m
1.33 m
1.26
1.88 m
5.54 brd (5.0) 3.84 m
1.36 m
1.16 m
1.38 m
1.17 m
0.98
a
a
0.97
1.02 brd
0.95 dd (24.7, 1.09
11.9)
0.70 brd (12.7)
(14.6)
a
8
9
1.65
1.66 m
1.55 m
2.18 brd (7.7) 2.43 m
2.41 m
1.36 dd (12.1,
5
.4)
1
1
0
1
a
2.57 dd (15.0, 2.60 dd (15.4, 2.75 m
.4) 8.6)
2.64 dd (18.0, 5.39
7.9)
2.37 m
1.42 m (2H)
1.47 m (2H)
2.51 dd (16.0,
9.0)
8
a
a
1.48
1.50
1.15 brd (16.0) 2.07 m
1.70 dd (15.4,
0.92 dd (16.1,
3.5)
6
.0)
a
a
a
1
2
4.06 m
4.08 m
5.11 m
4.24 brd
12.4)
2.21 d (17.6) 4.10 t (7.5)
1.55
1.63
4.83
(
1
1
1
3
4
5
1.92 m
1.38 m
9.95 s
1.40 m
9.93 s
1.88 dd (12.7, 1.91 m
.1)
1.88 m
1.63 m
1.84 m
1.72 m
2.04 m
2.62dd (13.4,
8.2)
1.78 m
8
1
.64 dd (11.2, 1.68 m
1.86 dd (12.2, 2.35 m
6.7)
1.60 dd (13.6,
5.4)
5.4)
1
1
6
7
4.71 dd (14.7, 4.73 dd (14.8, 4.65 q (7.5)
.8) 7.5)
4.66 dd (14.5, 4.77 dd (14.9, 4.84 dd (14.4,
4.57 dd (13.5,
8.0
7
7.4)
7.6)
7.7)
a
1.90 dd (15.2, 1.93 m
.0)
1.80 m
1.80 m
1.62 m
1.99 t (8.5)
2.76 d (5.7)
2.75 d (5.2)
1.92
7
1
1
8
9
1.39 s
1.39 s
1.29 s
1.05 s
0.92 s
0.79 s
1.44 s
1.51 s
1.52 s
1.02 s
0.72 d (4.1)
0.66 d (3.7)
0.31 d (3.9)
2.12 m
0.60 d (3.0)
0.24 d (3.6)
1.97 m
0.91 d (4.1)
0.20 d (4.4)
0.60 d (4.2)
0.60 d (4.3)
0.36 d (3.9)
0.47 d (4.1)
−0.04 d (4.2) −0.03 d (4.5) 0.00 d (4.2)
20
2.13 m
2.00 m
1.97 m
2.18 brd (7.7) 2.04 dd (12.3, 2.06 m
.9)
1.78 m
5
2
2
1
2
1.46 d (6.5)
1.48 d (6.5)
1.02 d (6.4)
1.44 d (6.4)
0.98 d (6.5)
1.54 d (6.4)
0.98 d (6.3)
0.99 d (6.0)
0.74 d (6.4)
2.85 dd (13.8, 2.85 dd (13.8, 2.77 m
2.75 dd (13.8, 2.78 dd (13.8, 2.88 dd (13.8, 2.55 dd (14.3, 2.04 m
2.5)
2.23 dd (14.7,
3.3)
2.7)
2.3)
2.2)
2.7)
1.73 brd (13.3) 1.79 m
5.01 d (11.1) 5.04 d (11.5)
5.2)
1.70 m
1.70 m
1.61 m
1.59 m
1.58 m
1.87 m
2.03 t (14.1)
23
24
25
26
4.60 d (6.1)
4.32 d (8.6)
4.59 brs
4.58 d (5.5)
4.29 d (8.6)
4.52 brd (4.0) 4.62 s
4.63 s
3.70 s
3.73 s
4.33d (8.6)
4.24 brd
12.4)
4.34 d (8.6)
4.34 brd (8.4) 1.58 s
4.10 d (8.4)
1.55 s
(
4.10 d (8.6)
4.11 d (8.6)
1.76 s
4.07 d (8.6)
1.75 s
4.00 d (8.6)
1.66 s
4.09 d (8.6)
1.77 s
2
2
2
3
3
1
2
3
7
1.76 s
0.82 s
0.98 s
1.12 s
1.78 s
1.08 s
1.00 s
1.33 s
1.63 s
1.69 s
0.93 s
1.21 s
1.59 s
1.60 s
0.97 s
1.23 s
8
0.88 s
0.86 s
0.85 s
0.93 s
0.61 s
0.80 s
0.96 s
9
1.05 s
1.04 s
0.94 s
1.12 s
0
1.22 s
1.20 s
1.12 s
1.21 s
-sugar
′
′
′
4.80 d (7.2)
4.48 t (7.5)
4.19 dd (9.0,
4.83 d (7.2)
4.49 t (8.0)
4.90 d (7.6)
4.06 t (8.1)
4.20 t (8.8)
4.19 dd (7.9,
2.9)
3
.2)
4
′
′
4.35 brs
4.35 brs
4.26 m
5
4.32 brd (11.2, 4.37 brd
.4) (12.9)
4.43 dd (11.2,
5.2)
2
3
.83 brd (10.9) 3.87 d (11.6) 3.83 t (10.7)
AcO-12
2.09 s
1.90 s
C
dx.doi.org/10.1021/np4000262 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 1. continued
a
b
c
Signals overlapped. Recorded at 500 MHz in pyridine-d . Recorded at 600 MHz in pyridine-d
5
5.
Table 2. 13C NMR Data of Compounds 1−9 (δ in ppm)
a
a
a
b
a
b
a
a
a
position
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
33.30 CH2
37.40 CH2
214.97 C
50.07 C
31.98 CH2
30.82 CH2
77.52 CH
40.71 C
32.24 CH2
31.11 CH2
77.73 CH
41.01 C
36.59 CH2
29.11 CH2
78.42 CH
39.96 C
36.39 CH2
28.62 CH2
78.14 CH
39.36 C
31.72 CH2
30.14 CH2
88.51 CH
41.35 C
30.49 CH2
29.44 CH2
87.93 CH
41.31 C
30.42 CH2
29.49 CH2
88.00 CH
41.30 C
31.06 CH2
30.48 CH2
77.75 CH
41.00 C
48.04 CH
21.00 CH2
25.64 CH2
45.64 CH
21.71 C
46.69 CH
20.54 CH2
25.57 CH2
45.38 CH
20.51 C
46.98 CH
20.81 CH2
25.97 CH2
46.01 CH
20.82 C
51.53 CH
19.10 CH2
27.07 CH2
134.31 C
137.56 C
37.98 C
49.85 CH
23.47 CH2
121.49 CH
146.67 C
142.07 C
37.94 C
45.97 CH
31.78 CH2
69.86 CH
53.02 CH
22.03 C
43.93 CH
18.39 CH2
21.44 CH2
38.52 CH
20.81 C
44.15 CH
18.55 CH2
21.68 CH2
38.70 CH
20.79 C
46.93 CH
20.70 CH2
25.80 CH2
46.16 CH
20.16 C
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
1
2
3
4
5
0
26.34 C
26.61 C
27.06 C
27.24 C
25.54 C
25.71 C
27.24 C
1
40.58 CH2
72.14 CH
50.46 C
40.25 CH2
72.08 CH
50.15 C
36.98 CH2
77.30 CH
47.85 C
35.21 CH2
71.38 CH
49.67 C
116.44 CH
38.11 CH2
43.44 C
40.63 CH2
72.26 CH
51.09 C
26.96 CH2
32.63 CH2
47.32 C
26.95 CH2
32.64 CH2
47.20 C
36.61 CH2
76.79 CH
48.35 C
2
3
4
47.59 C
47.51 C
48.84 C
50.55 C
48.26 C
48.57 C
55.34 C
55.30 C
48.69 C
5
44.22 CH2
72.84 CH
57.01 CH
12.97 CH3
29.07 CH2
26.37 CH
21.75 CH3
37.47 CH2
110.87 C
83.67 CH
79.78 C
43.96 CH2
72.61 CH
56.73 CH
12.61 CH3
29.34 CH2
26.09 CH
21.52 CH3
37.09 CH2
110.62 C
83.32 CH
79.54 C
43.80 CH2
72.53 CH
56.45 CH
13.61 CH3
30.04 CH2
26.13 CH
21.15 CH3
37.33 CH2
110.58 C
83.85 CH
79.68 C
41.04 CH2
73.68 CH
55.58 CH
12.10 CH3
19.84 CH3
27.07 CH
22.73 CH3
37.68 CH2
111.29 C
84.38 CH
80.22 C
40.63 CH2
72.68 CH2
55.25 CH
18.68 CH3
23.08 CH3
26.41 CH
20.83 CH3
37.04 CH2
110.97 C
83.92 CH
79.70 C
45.95 CH2
73.71 CH
56.96 CH
12.78 CH3
27.42 CH2
26.78 CH
22.54 CH3
38.02 CH2
111.31 C
84.33 CH
80.22 C
207.56 C
175.02 C
55.19 CH
18.03 CH3
21.83 CH2
28.49 CH
24.82 CH3
36.74 CH2
78.57 CH
79.88 CH
72.45 C
207.38 C
174.86 C
55.49 CH
18.21 CH3
22.07 CH2
28.48 CH
24.80 CH3
36.71 CH2
78.57 CH
79.86CH
72.43 C
43.88 CH2
80.46 CH
53.77 CH
13.29 CH3
29.86 CH2
26.90 CH
21.99 CH3
38.63 CH2
173.60 C
6
7
8
9
0
1
2
3
4
5
6
77.19 CH2
20.83 CH3
19.58 CH3
20.78 CH3
22.58 CH3
76.97 CH2
20.46 CH3
19.37 CH3
14.48 CH3
25.93 CH3
77.13 CH2
20.88 CH3
19.71 CH3
14.81 CH3
26.19 CH3
77.84 CH2
21.40 CH3
25.44 CH3
16.86 CH3
29.11 CH3
77.03 CH2
20.87 CH3
25.83 CH3
16.69 CH3
28.89 CH3
77.49 CH2
21.38 CH3
19.81 CH3
15.58 CH3
26.24 CH3
26.00 CH3
28.49 CH3
14.69 CH3
14.46 CH3
25.35 CH3
26.02 CH3
28.98 CH3
14.73 CH3
14.38 CH3
25.35 CH3
7
8
19.63 CH3
14.72 CH3
26.15 CH3
9
0
-sugar
′
′
′
′
″
108.06 CH
73.43 CH
75.17 CH
70.15 CH
67.46 CH2
107.52 CH
72.99 CH
74.68 CH
69.66 CH
67.15 CH2
107.48 CH
75.60 CH
78.61 CH
71.31 CH
67.14 CH2
AcO-12
170.57 C
170.65 C
2
1.66 CH3
21.42 CH3
a
b
Recorded at 125 MHz in pyridine-d . Recorded at 150 MHz in pyridine-d .
5
5
respectively. These observations could be explained by the
conjugated double bonds located at C-7/C-8 and C-9/C-11
1
1
and absence of an OH group at C-12. In the H− H COSY
spectrum, the correlations of the proton signals at δ 1.51 and
H
0
.77 (H-6) with the olefinic signal at δ 5.54 (H-7) and of the
H
proton signals at δH 2.12 and 1.92 (H-12) with the olefinic
signal at δH 5.54 (H-11) further supported this deduction.
Accordingly, the structure of 5 was determined as shown.
Yunnanterpene F (6) gave the molecular formula C H O
56 11
from its HREIMS data at m/z 652.3833 [M] . The H NMR
spectrum of compound 6 showed signals for a cyclopropane
1
1
Figure 1. Major HMBC (→), H− H COSY (−), and ROESY (↔)
correlations of compound 1.
3
5
+
1
−1
methylene at δ 0.20 (1H, d, J = 4.1 Hz) and 0.91 (1H, d, J =
bonds (λmax 242 nm; νmax 1631 cm ). The NMR spectroscopic
data of 5 closely resembled those of 4, except for the presence
of four olefinic carbon signals at δ 116.44, 121.49, 142.07, and
H
4
.4 Hz), six methyl groups at δ 1.00−1.78, and an anomeric
H
proton resonance at δ 4.80 (d, J = 7.2 Hz), suggesting that 6
C
H
146.67 and the absence of resonances of two methylenes and
was a 9,19-cycloartane-type triterpene glycoside. An HMBC
the presence of a hydroxymethine due to C-7, C-11, and C-12,
correlation between the anomeric proton at δ 4.80 (d, J = 7.2
H
D
dx.doi.org/10.1021/np4000262 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 2. X-ray crystal structures of 1 and 7.
Hz) and the methine signal at δ 88.51 (C-3) located the sugar
Cimilactone C (9) was determined to have the molecular
C
+
moiety at C-3. The sugar obtained after acid hydrolysis was
identified as L-arabinose by comparing its TLC and specific
rotation with a standard. The NMR data (Tables 1 and 2) of
the aglycone part of 6 resembled those of 2 with the exception
of an additional OH group, which was assigned to C-7 on the
basis of the correlations of H-6 (δ 2.04 and 1.33) and H-8 (δ
formula C H O by HREIMS (m/z 458.3027, [M] ). The IR
2
8
42
5
−1
spectrum of 9 showed absorptions for OH (3485 cm ) and
−
1
1
carbonyl (1729 and 1705 cm ) groups. H NMR signals for a
cyclopropane methylene at δ 0.36 (1H, d, J = 3.9 Hz) and
0.00 (1H, d, J = 4.2 Hz), an acetyl methyl group at δ 1.90, four
singlet methyl groups at δ 0.61, 0.80, 0.96, and 1.02, and a sec-
H
H
H
H
H
1
1
methyl group at δ 0.74 (d, J = 6.4 Hz) were observed. Analysis
2
.18) with the hydroxymethine proton (δ 3.84) in the H− H
COSY spectrum. In the ROESY spectrum, correlations of H-3/
H
H
of the data obtained led to the conclusion that 9 was a
tetranortriterpenoid with an acetoxy group. 13C NMR and
DEPT spectroscopic data of 9 were identical to the aglycone
H-5 and CH -28/H-7 suggested the β-orientation of the
3
substituents at C-3 and C-7. Therefore, the structure of 6 was
determined as shown.
Compounds 7 and 8 (15,16-seco-cimiterpenes A and B)
24
resonances of cimilactone A except for an upfield shift of the
C-3 signal by 9.95 ppm, which was consistent with the absence
of a sugar unit at C-3. Significant ROESY correlations of H-3/
H-5, H-12/CH -28, H-16/CH -28, and H-17/CH -28 sug-
both had the molecular formula C H O , as determined by
3
5
56 10
HREIMS. The NMR data of 7 were similar to the resonances of
5,16-seco-14-formyl-16-oxo-7-en-hydroshengmanol-3-O-β-D-
3
3
3
gested the β-orientation of the substituents at C-3, C-12, C-16,
and C-17. Therefore, the structure of 9 was established as
shown.
1
2
3
xylopyranoside, with the major differences at ring B and the
sugar unit. In the C spectrum of 1, a pair of double-bond
signals due to C-7 at δ 122.2 (d) and C-8 at δ 140.4 (s) were
1
3
1
0
The known compounds 12β-hydrocimilactone (10),
C
C
11
12
cimigenol (11), 12β-O-acetylcimigenol (12), 12β-hydro-
cimigenol (13), 12β-hydrocimigenol-7(8)-en (14), 12β-
hydrocimigenol-7(8)-3-one (15), 24-epi-7(8)-en-cimigenol-
-O-α-L-arabinopyranoside (16), 24-epi-7(8)-en-cimigenol-3-
absent, showing instead a methylene and a methine at δ 21.44
C
13
7
and 38.52, respectively. The sugar obtained after acid hydrolysis
was identified as L-arabinose by comparing its TLC and specific
14
15
3
rotation with a standard. In the ROESY spectrum, CH -18
3
15
O-β-D-xylosepyranoside (17), 7(8)-en-cimigenol-3-O-α-L-ara-
revealed a correlation with H-8, while CH -28 showed a
16
17
3
binopyranoside (18), 25-anhydrocimigenol (19),6 25-anhy-
correlation with H-7α, suggesting that CH -18 was β-orientated
3
drocimigenol-3-O-α-L-arabinopyranoside (20), acerinol
and that CH -28 was α-orientated. However, it was difficult to
18
19
20
3
(
21), acteol (22), 12β-O-acetylcimiracemonol (23), and
identify the stereochemistry of C-17, C-20, C-23, and C-24 by
ROESY experiments, due to the cleavage between C-15 and C-
21
dahurinol (24) were identified (see Supporting Information
for structures of 10−24) by comparing their physical and
spectroscopic data with reported data.
16. To confirm the structure and determine the absolute
configuration of the chiral centers, 7 was crystallized from
MeOH to afford a crystal of the orthorhombic space group
P2 2 2 , which was analyzed by X-ray crystallography. On the
All isolated compounds (1−24) were evaluated for their
−/−
inhibitory activities against the WT MEF, p53 +H-RasV12,
−/−
N236S
1
1 1
and p53 +p53
screening active structures targeting p53
Compounds 4 and 5 showed the most potent activities against
+H-RasV12 cells, a featured approach for
basis of 10 oxygen atoms in the molecule, the final refinement
of the Cu Kα data resulted in a Flack parameter of 0.06(10),
which allowed unambiguous assignment of the absolute
configuration (Figure 2). According to the projection, the
absolute configurations of C-17, C-20, C-23, and C-24 were
assigned as R, R, R, and S. Accordingly, the structure of 7 was
determined as shown. The NMR data of 8 were quite similar to
N236S
mutation.
−/−
N236S
the p53 +p53
+H-RasV12 cells, having IC values of 5.5
50
and 8.6 μM, respectively (Supporting Information, Table 1S).
However, 5 indicated nonselective cytotoxicity against the WT
MEFs, with an IC50 value of 6.0 μM. Compound 4 exhibited
approximately 3-fold higher selectivity against the WT MEFs
cells (IC 14.5 μM) than 5. Thus, the mechanism of action of
those of 7 except for the sugar moiety at δ 107.48 (d), 75.60
C
50
(
d), 78.61 (d), 71.31 (d), and 67.14 (t). The sugar obtained
compound 4 is worth studying in a more advanced way.
Compounds 9, 14, 19, 21, and 22 showed greater selective
activities against the p53− +H-RasV12 cells (IC 7.5, 9.8, 6.2,
after acid hydrolysis was identified as D-xylose by comparing its
TLC and specific rotation with a standard. On the basis of the
ROESY correlations of H-17 and Me-21/H-22α, H-23/H-22β,
and H-23/H-24, identical NMR data, and the coupling
constants of H-24, the same configurations of C-17 (R), C-20
/−
5
0
10.2, and 7.4 μM) than against the WT MEFs and
−
/−
N236S
p53 +p53
+H-RasV12 cell line. The phenomenon may
N236S
be due to the following: (1) p53
can interact with H-
RasV12 in the p53− +p53
/−
N236S
+H-RasV12 cells and facilitate
(R), C-23 (R), and C-24 (S) of 7 and 8 can be concluded.
Thus, the structure of 8 was determined as shown.
cell proliferation; (2) the normal function of P53 in the WT
E
dx.doi.org/10.1021/np4000262 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
MEFs can regulate the cellular stress response to noxious
signals and promote cell growth.
(3β,12β,23S,24R,25S)-16,23:23,26-Diepoxy-3,12,24,25-tetrahy-
droxy-9,19-cycloartane (2): white powder; [α]2 −89.3 (c 0.08,
7
D
MeOH); IR (KBr) ν 3422, 2953, 2871, 1447, 1379, 1165, 1089,
max
−1
1
13
1
020, 957 cm ; H (C D N, 500 MHz) and C NMR (C D N, 125
5
5
5
5
+
EXPERIMENTAL SECTION
MHz) spectra see Tables 1 and 2; ESIMS m/z 527 [M + Na] ;
HREIMS m/z 504.3444 (calcd for C H O , 504.3451).
■
General Experimental Procedures. Optical rotations were
measured in MeOH with a Horiba SEAP-300 polarimeter. H and
C NMR spectra were recorded in pyridine-d on Bruker DRX-500
30 48
6
1
(3β,12β,23S,24R,25S)-16,23:23,26-Diepoxy-12-acetoxy-3,24,25-
trihydroxy-9,19-cycloartane (3): white powder; [α]2
MeOH); IR (KBr) ν 3448, 2935, 2873, 1715, 1455, 1382, 1248,
7
−112.1 (c 0.09,
1
3
D
5
max
and Avance III-600 MHz spectrometers (Bruker, Zu
̋
rich, Switzerland).
−1
1
13
1
095, 983 cm ; H (C D N, 500 MHz) and C NMR (C D N, 125
5
5
5
5
Unless otherwise specified, chemical shifts (δ) were expressed in ppm
with respect to the solvent signals. Mass spectra were performed on a
VG Autospec-3000 spectrometer. Infrared spectra were recorded on a
Shimadzu IR-450 instrument with KBr pellets. Thin-layer chromatog-
raphy was performed on precoated TLC plates (200−250 μm
+
MHz) spectra see Tables 1 and 2; ESIMS m/z 569 [M + Na] ;
HREIMS m/z 546.3557 (calcd for C H O , 546.3557).
32
50
7
(
3β,12β,23S,24R,25S)-16,23:23,26-Diepoxy-3,12,24,25-tetrahy-
27
droxylanosta-8(9)-ene (4): white powder; [α] −71.1 (c 0.05,
D
MeOH); IR (KBr) ν 3452, 2961, 2874, 1630, 1453, 1377, 1168,
1034, 961 cm ; H (C D N, 500 MHz) and C NMR (C D N, 150
MHz) spectra see Tables 1 and 2; ESIMS m/z 527 [M + Na] ;
HREIMS m/z 504.3454 (calcd for C H O , 504.3451).
max
thickness, silica gel 60 F2 , Qingdao Marine Chemical, Inc.), and
−1
1
13
54
5
5
5
5
spots were visualized by heating after spraying with 10% aqueous
+
H SO . Semipreparative HPLC was performed on an Agilent 1100
2
4
30
48
6
liquid chromatograph with a YMC-Pack Pro C18 RS 10 mm × 250
mm column. Silica gel (200−300 mesh, Qingdao Marine Chemical,
Inc.), Lichroprep RP-18 (40−63 μm, Merck), and Sephadex LH-20
(
3β,23S,24R,25S)-16,23:23,26-Diepoxy-3,24,25-trihydroxylanos-
27
ta-7(8),9(11)-diene (5): white powder; [α] −136.8 (c 0.05, MeOH);
D
UV (MeOH) λ (log ε) 242 (0.51), 196 (0.19). IR (KBr) ν 3425,
max
max
−1 1
(
(
20−150 μm, Pharmacia) were used for column chromatography
CC).
2958, 2874, 1631, 1452, 1374, 1156, 1033, 948 cm ; H (C D N, 500
5 5
MHz) and 13C NMR (C D N, 125 MHz) spectra see Tables 1 and 2;
5
5
+
Plant Material. The aerial parts of Cimicifuga yunnanensis (1.5 kg)
ESIMS m/z 509 [M + Na] ; HREIMS m/z 486.3344 (calcd for
were collected from Daocheng County, Sichuan Province, China, in
September 2008 and were identified by Prof. Shengji Pei, Kunming
Institute of Botany, Chinese Academy of Sciences. A voucher
specimen (KUN No. 200809007) has been deposited at the State
Key Laboratory of Phytochemistry and Plant Resources in West
China, Kunming Institute of Botany, Chinese Academy of Sciences,
China.
Extraction and Isolation. The dried and milled aerial parts of C.
yunnanensis (1.5 kg) were extracted with MeOH (3 × 5 L; 24 h each)
at room temperature to give a residue (137.8 g) after evaporating
under vacuum at 50 °C. The extract was subjected to silica gel CC (3
C H O , 486.3345).
(3β,6β,12β,23S,24R,25S)-16,23:23,26-Diepoxy-6,12,24,25-tetrahy-
droxy-9,19-cycloart-3-O-β-D-xylopyranoside (6): white powder; [α]
30
46
5
2
7
D
−
21.6 (c 0.10, MeOH); IR (KBr) ν 3441, 2948, 2874, 1711, 1456,
383, 1142, 1085, 1010 cm ; H (C D N, 600 MHz) and C NMR
max
−
1
1
13
1
5 5
(
C D N, 150 MHz) spectra see Tables 1 and 2; ESIMS m/z 675 [M +
5 5
+
Na] ; HREIMS m/z 652.3833 (calcd for C H O , 652.3823).
35 56 11
(
3β,17R,23R,24S)-15,16-seco-14-Formyl-16-oxo-16,23-epoxy-
9
2
2
,19-cycloart-3-O-α-L-arabinopyranoside (7): colorless crystals; mp
27
13-216 °C; [α]D −65.4 (c 0.05, MeOH); IR (KBr) ν 3457, 2939,
884, 1711, 1640, 1458, 1384, 1257, 1142, 1069, 995 cm ;
max
−1
1
H
1
3
(
C D N, 500 MHz) and C NMR (C D N, 125 MHz) spectra see
5
5
5
5
kg, 10 × 150 cm) and eluted with CHCl −MeOH [100:0 (3 L), 50:1
3
+
Tables 1 and 2; ESIMS m/z 659 [M + Na] ; HREIMS m/z 636.3871
(
6 L), 20:1 (8 L), 10:1 (5 L), 0:100 (4 L)] to afford fractions A (16.7
(
calcd for C H O , 636.3873).
35 56 10
g), B (22.7 g), C (18.5 g), D (19.3 g), and E (20.7). Fraction B was
(
3β,17R,23R,24S)-15,16-seco-14-Formyl-16-oxo-16,23-epoxy-
27
divided into five subfractions (B.1−B.5) after performing RP-18 CC
9
,19-cycloart-3-O-β-D-xylosepyranoside (8): white powder; [α]
D
(
500 g, 6 × 50 cm), eluting with MeOH−H O (gradient from 50:50
2
−
66.0 (c 0.06, MeOH); IR (KBr) ν
640, 1460, 1378, 1239, 1070, 9789 cm ; H (C D N, 500 MHz) and
3424, 2962, 2870, 1711,
max
−1
to 100:0). Fraction B.3 (4.6 g) was subjected to repeated silica gel CC
1
1
5 5
(
1
60 g, 5 × 40 cm), eluted with CHCl −Me CO (gradient from 20:1 to
13
3
2
C NMR (C D N, 125 MHz) spectra see Tables 1 and 2; ESIMS m/z
5
5
0:1), and then repeated semipreparative HPLC (eluted with
+
6
6
59 [M + Na] ; HREIMS m/z 636.3876 (calcd for C H O ,
36.3873).
35 56 10
CH CN−H O, gradient from 60:40 to 85:15) to yield 1 (6.0 mg),
3
2
2
(7.2 mg), 3 (6.9 mg), 12 (4.8 mg), 13 (3.2 mg), 15 (4.3 mg), and 19
4.7 mg). Compounds 11 (4.8 mg), 14 (3.5 mg), 21 (4.3 mg), 22 (5.7
mg), 23 (3.5 mg), and 24 (3.6 mg) were isolated from fraction B.4
3.8 g) by conducting silica gel CC (40 g, 5 × 40 cm), eluting with
CHCl −Me CO (20:1, 7 L), followed by repeated semipreparative
(
3β,12β,16β)-3-Hydroxy-12-acetoxy-24,25,26,27-tetranor-9,19-
(
27
cycloart-23,16-olide (9): white powder; [α]D −111.5 (c 0.12,
MeOH); IR (KBr) νmax 3485, 2924, 2860, 1729, 1705, 1439, 1378,
(
−1
1
13
1
262, 1126, 1031, 987 cm ; H (C D N, 500 MHz) and C NMR
5 5
3
2
(C D N, 125 MHz) spectra see Tables 1 and 2; ESIMS m/z 481 [M +
5 5
HPLC (eluted with CH CN−H O, gradient from 60:40 to 90:10).
+
3
2
Na] ; HREIMS m/z 458.3027 (calcd for C H O , 458.3032).
28 42 5
Fraction B.5 (3.9 g) was applied to a silica gel column (30 g, 4 × 40
cm), eluted with CHCl −Me CO (20:1, 8 L), then subjected to
X-ray Crystal Structure Analysis. Colorless crystals of 1 and 7
were obtained from CH OH. Intensity data were collected at 100 K on
a Bruker APEX DUO diffractometer equipped with an APEX II CCD,
using Cu Kα radiation. Cell refinement and data reduction were
performed with Bruker SAINT. The structures were solved by direct
methods using SHELXS-97. Refinements were performed with
SHELXL-97, using full-matrix least-squares, with anisotropic displace-
ment parameters for all the non-hydrogen atoms. The H atoms were
placed in calculated positions and refined using a riding model.
Molecular graphics were computed with PLATON. Crystallographic
data (excluding structure factor tables) for the structures reported have
been deposited with the Cambridge Crystallographic Data Center as
supplementary publications no. CCDC 918339 for 1 and CCDC
918340 for 7. Copies of the data can be obtained free of charge on
application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax:
Int. +44(0) (1223) 336 033; e-mail: deposit@ccdc.cam.ac.uk].
Yunnanterpene A (1): C H O , M = 502.67, orthorhombic, a =
3
2
3
semipreparative HPLC (eluted with CH CN−H O, gradient from
3
2
65:35 to 90:10) to afford 4 (3.7 mg), 5 (3.2 mg), 9 (5.9 mg), and 10
(
(
6.6 mg). Fraction C (18.5 g) was separated into four subfractions
C.1−C.4) by performing RP-18 CC (300 g, 5 × 40 cm), eluting with
2
5
MeOH−H O (gradient from 50:40 to 90:10). Fraction C.3 (2.3 g)
2
was subjected to silica gel CC (50 g, 4 × 40 cm) eluted with CHCl −
3
Me CO (gradient from 10:1 to 5:1), then repeated semipreparative
2
HPLC (eluted with CH CN−H O, gradient from 60:40 to 75:25) to
3
2
obtain 6 (4.5 mg), 16 (3.8 mg), 20 (3.3 mg), and 18 (2.8 mg).
Fraction C.2 (4.8 g) was subjected to silica gel CC (50 g, 4 × 40 cm),
eluting with CHCl −Me CO (10:1, 13 L), to yield 7 (7.2 mg), 8 (5.0
3
2
mg), and 18 (4.2 mg).
(
12β,23S,24R,25S)-16,23:23,26-Diepoxy-12,24,25-trihydroxy-
27
9
−
1
,19-cycloart-3-one (1): colorless crystals; mp 226−228 °C; [α]
D
71.7 (c 0.09, MeOH); IR (KBr) ν 3439, 2965, 2857, 1706, 1455,
416, 1343, 1172, 1020, 961 cm ; H (C D N, 500 MHz) and
max
30 46
6
−1
1
13
C
10.1345(2) Å, b = 15.4301(3) Å, c = 16.8872(3) Å, α = 90.00°, β =
5
5
3
NMR (C D N, 150 MHz) spectra see Tables 1 and 2; ESIMS m/z 525
90.00°, γ = 90.00°, V = 2640.76(9) Å , T = 100(2) K, space group
5
5
+
−1
[
M + Na] ; HREIMS m/z 502.3287 (calcd for C H O , 502.3294).
P2 2 2 , Z = 4, μ(Cu Kα) = 0.689 mm , 21 112 reflections measured,
30
46
6
1 1 1
F
dx.doi.org/10.1021/np4000262 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
4
0
789 independent reflections (Rint =2 0.0274). The final R values were
1
REFERENCES
■
.0282 (I > 2σ(I)). The final wR(F ) values were 0.0726 (I > 2σ(I)).
(
5
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2
The final R values were 0.0283 (all data). The final wR(F ) values
were 0.0726 (all data). The goodness of fit on F was 1.046. Flack
parameter = 0.06(11). The Hooft parameter is 0.02(3) for 2029
Bijvoet pairs.
1
2
(
(
1
5,16-seco-Cimiterpene A (7): C H O , M = 636.80, ortho-
35 56 10
Qiu, M. H.; Gross, M. L.; Qiu, S. X. Beilstein. J. Org. Chem. 2007, 3, 1−
rhombic, a = 8.3726(2) Å, b = 10.4589(2) Å, c = 36.9890(8) Å, α =
3
6.
9
0.00°, β = 90.00°, γ = 90.00°, V = 3239.06(12) Å , T = 100(2) K,
−1
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space group P2 2 2 , Z = 4, μ(Cu Kα) = 0.769 mm , 15 921
1
1 1
reflections measured, 5572 independent reflections (Rint = 0.0287).
The final R values were 0.0294 (I > 2σ(I)). The final wR(F ) values
were 0.0765 (I > 2σ(I)). The final R values were 0.0295 (all data).
The final wR(F ) values were 0.0766 (all data). The goodness of fit on
F was 1.063. Flack parameter = 0.06(10). The Hooft parameter is
0
2
1
1
2
2
(
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Hydrolysis and Identification of the Sugar Moieties in
(
Compounds 6−8. Compounds 6, 7, and 8 (4 mg of each) were
dissolved in MeOH (5 mL) and refluxed with 0.5 N HCl (3 mL) for 4
h. Each reaction mixture was diluted with H O and extracted with
CHCl (3 × 10 mL). Each aqueous layer was then neutralized by
Ag CO , and the precipitate filtered to give a monosaccharide. The
(
2
3
1
(
2
3
monosaccharide from compounds 6 and 7 had an R (EtOAc−
f
2
0
CHCl −MeOH−H O, 3:2:2:1) and specific rotation ([α] +82.8 (c
3
2
D
(
2
(
0.09, MeOH)) corresponding to those of L-arabinose (Sigma-Aldrich),
while the monosaccharide from compound 8 had an R (EtOAc−
f
2
0
CHCl −MeOH−H O, 3:2:2:1) and specific rotation ([α] +30.2 (c
3
2
D
0.13, H O)) corresponding to those of D-xylose (Sigma-Aldrich).
2
−
/−
(
4
(
Cytotoxicity Bioassay. WT MEF, p53 +H-RasV12 MEF, and
−/−
N236S
p53 +p53
+ H-RasV12 MEF cell lines were used in the
cytotoxic assay. Cells were cultured in DMEM medium (Hyclone,
USA), supplemented with 10% fetal bovine serum (Hyclone, USA), in
Chim. Acta 2009, 92, 112−120.
5
% CO at 37 °C. The assay was performed according to the MTT (3-
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2
(
4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method
26,27
in 96-well microplates.
Briefly, 100 μL of cells was seeded into
each well of 96-well cell culture plates and allowed to adhere for 12 h
before addition of test compounds, while suspended cells were seeded
just before drug addition with an initial density of 1 × 10 cells/mL.
Each tumor cell line was exposed to the test compound at
concentrations of 0.064, 0.32, 1.6, 8, and 40 μM in triplicates for 48
h, with cisplatin (Sigma, USA) as a positive control. After compound
treatment, cell viability was detected and a cell growth curve was
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5
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2
8
graphed. IC50 values were calculated by Reed and Muench’s method.
(21) Kusano, G.; Murakami, Y.; Sakurai, N.; Takemoto, T. Yakugaku
ASSOCIATED CONTENT
Zasshi 1976, 96, 82−85.
■
(
22) Chen, S. N.; Li, W. K.; Fabricant, D. S.; Santarsiero, B. D.;
*
S
Supporting Information
Mesecar, A.; Fitzloff, J. F.; Fong, H. H. S.; Farnsworth, N. R. J. Nat.
This material ( H, 13C, HSQC, HMBC, COSY, ROESY,
ESIMS, and HREIMS spectra of compounds 1−9, X-ray data of
compounds 1 and 7, structures of compounds 10−24, and
bioassay results of compounds 1−24) is available free of charge
via the Internet at http://pubs.acs.org.
1
Prod. 2002, 65, 601−605.
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2
003, 51, 1215−1216.
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(
̈
25) Sheldrick, G. M. SHELXL97; University of Gottingen:
AUTHOR INFORMATION
Corresponding Author
Tel: (86) 871-65223257. Fax: (86) 871-65223255. E-mail:
mhchiu@mail.kib.ac.cn.
Germany, 1997.
■
*
(
26) Mosmman, T. Immunol. Method 1983, 65, 55−63.
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Czerwinski, M. J.; Fine, D. L. Cancer Res. 1988, 48, 589−601.
(28) Reed, L. J.; Muench, H. Am. J. Hyg. 1938, 27, 493−497.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Natural
Science Foundation of China (No. U1132604), the Knowledge
Innovation Program of the CAS (Grant Nos. KZCX2-XB2-15-
0
3, KSCX2-EW-R-15), the Top Talents Program of Yunnan
Province (2009C1120), and the Foundation of State Key
Laboratory of Phytochemistry and Plant Resources in West
China (P2010-ZZ14).
G
dx.doi.org/10.1021/np4000262 | J. Nat. Prod. XXXX, XXX, XXX−XXX